The quantitative determination of analytes in biological fluids such as whole blood is of great importance in the diagnosing and treating of certain medical conditions. For example, determining the glucose level in the blood of diabetic individuals, who must frequently check the glucose level in their blood to regulate their diets and medication. Measuring the glucose content of blood can be done by several methods. One method employs an electrochemical biosensor. Another method provides a visual indication of the glucose content such as by developing a color by reaction of an indicator. The present invention is of the later type, although it may have application in electrochemical methods as well.
Electrochemical methods have been described in many patents. They may be divided into several categories including coulometic, amperometric, and cyclic voltammetry. One recent published U.S. patent application is 2003/0094384A1. Japanese published application 2000171428A2 describes the preparation of a glucose electrolytic sensor.
There have been many patents describing methods employing indicators that develop color or other measurable responses when they are chemically oxidized as the last step of a series of reactions. In general, such methods may be broadly divided into those that employ analyte oxidases (e.g., glucose oxidase) that those that employ analyte dehydrogenases (e.g., glucose dehydrogenase). The procedures used are similar, but they employ different enzymes, mediators and indicators.
Methods using glucose oxidase enzymes are taught in many US patents and patent applications. Representative are U.S. Pat. No. 4,211,845; U.S. Pat. No. 4,808,529; U.S. Pat. No. 5,116,729; U.S. Pat. No. 5,264,348; U.S. Pat. No. 5,620,863; and U.S. 2003/0077702 A1. These patents/patent application teach a method in which glucose is oxidized to gluconic acid with the release of hydrogen peroxide. The hydrogen peroxide is said to oxidize an indicator in the presence of a peroxidase to produce a measurable color, indicating the glucose content of the blood sample. Some recent patents suggest a process in which the glucose is converted first to gluconic acid and then to gluconolactone with the release of hydrogen peroxide. It has also been suggested that the gluconolactone is formed first and then hydrolyzed to gluconic acid. Regardless of which process scheme is correct, glucose oxidase enzymes have been used widely in dry strips and in other techniques for measuring the glucose contact of blood.
Various indicators have been employed in glucose sensors, such as benzidine-type indicators and heterocyclic azines. For example, 3,3′,5,5′-tetramethylbenzidine and syringaldazine, luminol, o-tolidine, o-dianisitine, among others. One family of indicators is that of tetrazolium dye precursors. Examples of patents describing such indicators include U.S. Pat. No. 5,126,275, U.S. Pat. No. 5,322,680, U.S. Pat. No. 5,300,637, U.S. Pat. No. 5,290,536, U.S. Pat. No. 5,360,595 and U.S. Pat. No. 6,586,199. Tetrazolium indicators are used in the invention to be described below.
Of particular interest with regard to the present invention is the method described in U.S. Pat. No. 6,200,773 and its parent U.S. Pat. No. 5,902,731. In these patents, a test of the glucose content of blood employs glucose dehydrogenase (GDH), with NAD or PQQ or their derivatives, as a cofactor, a tetrazolium dye precursor, a diaphorase enzyme or an analog, and a nitrite salt. FIG. 5 of the '773 patent is a diagram of the process by which glucose is detected by development of color from the reduction of the tetrazolium dye precursor to a formazan.
PQQ (pyrroloquinoline quinone or its derivatives or isomers) as a cofactor for glucose dehydrogenase (GDH) enzymes has been of recent interest, as is evident from the disclosures in U.S. 2002/0076751 A1; EP 1 167 519 A1; U.S. Pat. No. 6,103,509; and JP 11243949 A2. In general, these disclosures teach the genetic modification of glucose dehydrogenase enzymes that are said to provide improved performance in glucose sensors.
The present inventors unexpectedly have found that a process similar to that discussed in U.S. Pat. No. 6,200,773 can be carried out in the absence of a mediator, such as the diaphorase enzyme. This finding makes possible a simpler method of detecting glucose in blood samples, as will be seen in the description of the invention below.